Opioids are widely used analgesics; however prolonged use results in tolerance, a progressively decreasing responsiveness to the drug. Desensitization and internalization of u-opioid receptors (MOR) are important in the actions of opioids and appear to contribute to the development of tolerance. Desensitization and internalization have been well characterized in naive cells, but how these processes are regulated in neurons during chronic treatment is unclear. Knowing what cellular adaptations occur in response to chronic agonist exposure will better our understanding of the mechanisms contributing to tolerance. The purpose of this proposal is to investigate how regulation of u-opioid receptor desensitization and endocytosis are altered during chronic morphine treatment. Acute desensitization is an uncoupling of the receptor from G-protein activation that occurs during a sustained application of a high concentration of agonist. Agonist-bound receptors also undergo endocytosis. I hypothesize that an upregulation of agonist-induced internalization occurs in chronic-morphine treated animals, resulting in reduced recovery from desensitization. This hypothesis is based on studies examining alterations in the actions of opioids in rat locus coeruleus neurons following chronic-morphine treatment. Agonist efficacy was decreased, desensitization was facilitated, and recovery was incomplete in LC neurons from chronic-morphine-treated rats. To test my hypothesis, I will record MOR coupling to activation of potassium channels in locus coeruleus (LC) neurons from mice expressing a FLAG-tagged u-opioid receptor (FLAG-MOR). This will allow us to study MOR coupling and trafficking simultaneously in a live brain slice. The first goal of this project is to characterize activation, desensitization, and internalization of FLAG-MOR to [Met]5-enkephalin in naTve cells using a combination of electrophysiology and imaging. The second goal is to treat mice chronically with morphine and determine what role internalization plays in the reduced recovery from desensitization that is observed. Understanding the cellular mechanisms of MOR regulation during chronic exposure to morphine will provide insight into the processes that lead to the development of tolerance to opioids. This understanding may allow for the development of effective analgesics which do not produce tolerance. [unreadable] [unreadable] [unreadable]